Combination Therapy

ABSTRACT

The invention relates to producing humoral response to P2X7 receptors in individuals having cancer, and to minimizing the progression of cancer in said individuals.

FIELD OF THE INVENTION

The invention relates to a method of treating or ameliorating diseasesthat are associated with non-functional P2X₇ receptor expression,including cancer.

BACKGROUND OF THE INVENTION

Purinergic (P2X) receptors are ATP-gated cation—selective channels. Eachreceptor is made up of three protein subunits or monomers. To date sevenseparate genes encoding P2X monomers have been identified: P2X₁, P2X₂,P2X₃, P2X₄, P2X₅, P2X₆, P2X₇.

P2X₇ receptors are of particular interest as the expression of thesereceptors is understood to be limited to cells having potential toundergo programmed cell death, such as thymocytes, dendritic cells,lymphocytes, macrophages and monocytes. There is some expression of P2X₇receptors in normal homeostasis, such as on erythrocytes and in othercell types such as skin at generally lower levels.

Interestingly, a P2X₇ receptor containing a monomer having a cisisomerisation at Pro210 (according to SEQ ID NO: 1) and which hascompromised ATP binding function at the affected site or sites has beenfound on cells that are understood to be unable to undergo programmedcell death, such as pre-neoplastic cells and neoplastic cells. Thisisoform of the receptor has been referred to as a “non-functional”receptor and describes a form of the receptor unable to extend theoperating non-selective calcium channel into an apoptotic pore.

Antibodies generated from immunisation with a peptide including Pro210bind to non-functional P2X₇ receptors at the altered ATP binding site/sformed between adjacent monomers. However, they do not bind to P2X₇receptors capable of binding ATP at any of the three available sites.Accordingly, these antibodies are useful for selectively detecting manyforms of carcinoma and haemopoietic cancers and to treatment of some ofthese conditions.

WO02/057306A1 and WO03/020762A1 discuss a probe in the form of amonoclonal antibody for distinguishing between functional P2X₇receptors, defined as those receptors able to form a non-selective Ca/Nachannel that is additionally able to form an apoptotic pore uponextended binding of ATP, and non-functional P2X₇ receptors, defined asthose receptors able to form the non-selective channel but that areunable to extend opening of the channel to an apoptotic pore.

WO2009/033233 discusses an epitope exposed on non-functional receptorsbut not functional receptors and antibodies for binding thereto.

There exists a need for an alternative or improved treatment of diseasescaused by or associated with non-functional P2X₇ receptor expression,such as cancer.

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

SUMMARY OF THE INVENTION

In certain embodiments there is provided a method for minimising theprogression of cancer in an individual who has received a non selfantigen binding site for treatment of the cancer, the method includingthe steps of:

-   -   providing an individual who has received a non self antigen        binding site for treatment of the cancer;    -   forming an immune response in the individual to a P2X₇ receptor;

thereby minimising the progression of cancer in the individual.

In one form of the above described method, the individual may not havedetectable non self antigen binding sites in circulation at the timethat the immune response is formed in the individual.

Further, the individual may not have detectable cancer at the time thatthe immune response is formed in the individual, for example, the cancermay have substantially diminished in size, mass or other physicalmeasure as a consequence of administration of an antigen binding site tothe individual at the time that the immune response is formed,particularly to a non functional P2X7 receptor, or to acancer-associated P2X7 receptor, in the individual.

The immune response may be formed by an immunogen. The immunogen may beprovided in the form of a P2X₇ receptor, or a fragment of a P2X₇receptor that is capable of inducing an immune response to a P2X₇receptor in the individual.

The immunogen may contain at least one sequence that is capable of beingpresented on a major histocompatibility complex class II molecule and/oris capable of interacting with a T or B-cell receptor or a B-cellmembrane bound-immunoglobulin.

According to the invention, the individual is human, in which case theimmunogen is provided in the form of a human P2X₇ receptor, or fragmentthereof that is capable of inducing an immune response to a P2X₇receptor.

Typically the immune response that is formed in the individual isspecific for non-functional P2X₇ receptors, in which case antibodies orcellular components that are reactive with non-functional P2X₇ receptors(i.e. with one or more sites unable to bind ATP), but not reactive withfunctional P2X₇ receptors (i.e. ATP binding receptors) are formed in theindividual.

In a preferred form, the immunogen is provided in an initialadministration to the individual, thereby forming a response thatincludes IgM production.

In a further preferred form, the immunogen, which has been provided inan initial administration to the individual, thereby forming a responsethat includes IgM production, is administered at a later time, in afurther administration to the initial administration, thereby forming aresponse that includes IgG production. In this embodiment, typically thefurther administration of immunogen occurs when the level of IgM incirculation in the individual is substantially undetectable.

The immune response may be a humoral and/or cellular response.

A humoral response may include the transformation of B-cells into plasmacells, which secrete antibody, Th2 activation and cytokine production,germinal centre formation and isotype switching, affinity maturation ofB-cells and/or memory cell generation.

A cellular response may include activating antigen-specific cytotoxicT-lymphocytes, activating macrophages and natural killer cells and/orstimulating cells to secrete cytokines.

The humoral and/or cellular response formed in the individual may treator ameliorate a cancer in the individual, or minimise the progression ofcancer in the individual.

In the above described embodiments, the antigen binding sites receivedby the individual may be reactive with any biomarker that is associatedwith cancer. Examples include antigen binding sites against P2X₇,especially, non-functional P2X₇, against VEGF, especially VEGF A, C orD, Her-2, CD20 or others. Typically the antigen binding sites receivedby the individual are reactive with P2X₇ receptor, especially anon-functional P2X₇ receptor.

In another embodiment there is provided a use of a P2X₇ receptor orfragment thereof in the manufacture of a medicament for the treatmentof, or for the inhibition of progression of cancer in an individual whohas received an anti-P2X₇ receptor antigen binding site for treatment ofthe cancer.

In another embodiment there is provided a composition for treating, orfor inhibiting the progression of a cancer in a human including a P2X₇receptor or fragment thereof. Preferably the composition furtherincludes a carrier, excipient or diluent. Preferably, the compositionfurther includes an adjuvant.

In another embodiment there is provided a composition for use in thetreatment, or for use in the inhibition, of the progression of a cancerin a human including a P2X₇ receptor or fragment thereof. Preferably thecomposition further includes a physiologically acceptable carrier,excipient or diluent. Preferably, the composition further includes anadjuvant.

In another embodiment, there is provided a composition when used in amethod of the invention.

In a preferred form, the composition enables the formation of a primaryimmune response (including IgM production) upon initial administrationof the immunogen to the individual, and a second immune response(including IgG production) upon administration of the immunogen furtherto the initial administration.

In other embodiments there is provided a kit or composition fortreatment of, or for inhibiting the progression of a cancer in a human,the kit including:

-   -   an immunogen capable of causing the formation of an immune        response to a human P2X₇ receptor when administered to a human;    -   an antigen binding site that binds to a biomarker associated        with cancer; and    -   written instructions for use in a method described above.

In one preferred form, the antigen binding site provided in the kit isreactive with a P2X₇ receptor, preferably a non-functional P2X₇receptor.

Without being bound by any theory or mode of action, it is believed thatthe present invention provides an alternative and/or improved treatmentregime for the reason that endogenous immune components such asantibodies and antigen specific cells that arise from immunisation havea longer and greater exposure to cell surface P2X₇ receptors, after theadministration of antigen binding sites has been completed and thecirculating level of non self anti-P2X₇ antigen binding sites has becomeundetectable.

Further, it is believed that P2X₇ receptor crowding, as arises when highconcentrations of non self or exogenous antibodies are provided in anindividual, minimises the level of specific antibody binding to the keyP2X₇ epitopes that provide for an anti cancer immune response, therebylimiting the efficacy of immunotherapy. The inventors have found that anantibody response arising from immunisation of an individual withimmunogen according to the invention described herein provides for anamount, titre or concentration of antibody that does not cause receptorcrowding, thereby improving the efficacy of immunotherapy, particularlyat a time when cancer in the individual may be substantiallyundetectable.

In one embodiment there is provided a process for forming a humoralimmune response to cancer-associated P2X₇ receptors in an individual whohas received an anti-cancer antigen antibody for therapy of cancer,including the steps of:

-   -   forming an immune response in the individual to an immunogen in        the form of a cancer-associated P2X₇ receptor or fragment        thereof;

wherein the immune response is formed in the individual at a time whereanti-cancer antigen antibody administered for treatment of cancer is ata level or concentration that is substantially undetectable in theindividual; and/or

the humoral immune response to cancer-associated P2X₇ receptors isformed according to an immunisation schedule whereby the amount ofantibody formed in the individual to cancer-associated P2X₇ receptor isabout 0.1 to 25 mg/kg individual.

In another embodiment there is provided a composition for use in forminga humoral immune response to cancer-associated P2X₇ receptors in anindividual who has received an anti-cancer antigen antibody for therapyof cancer, said composition including an immunogen in the form of acancer-associated P2X₇ receptor or fragment thereof. Preferably theimmune response is formed in the individual at a time where anti-cancerantigen antibody administered for treatment of cancer is at a level orconcentration that is substantially undetectable in the—individual;and/or the humoral immune response to cancer-associated P2X₇ receptorsis formed according to an immunisation schedule whereby the amount ofantibody formed in the individual to cancer-associated P2X₇ receptor isabout 0.1 to 25 mg/kg individual.

Further aspects of the present invention and further embodiments of theaspects described in the preceding paragraphs will become apparent fromthe following description, given by way of example and with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

Patient with moderate tumor burden immunized with peptideGHNYTTRNILPGLNITC coupled to KLH carrier in presence of adjuvant. TheELISA plate carries the peptide and the serum is serially diluted todetect for the presence of antibodies to the peptide. Pre-immune serum(blue), serum obtained immediately pre-boost at Week 4 (red) and serumat Week 13 (9 weeks post boost, green). No antibodies were detected inthe pre-boost serum taken 4 weeks post initial immunisation. This isbelieved to be a consequence of existing tumour mass that binds antibodyarising from the immunisation. The Week 13 sample shows a small level ofcirculating anti-nfP2X₇ antibody response as the tumour burden wasdecreased. Intermediate measurements taken from Weeks 5 to 12 showedlittle circulating antibody was present in serum

FIG. 2

Patient with very small residual tumor burden immunized with peptideGHNYTTRNILPGLNITC coupled to KLH carrier in presence of adjuvant. TheELISA plate carries the peptide and the serum is serially diluted todetect for the presence of antibodies to the peptide. Pre-immune serum(blue), serum obtained immediately pre-boost at Week 4 (red) and at Week5 (1 week post boost, green). Since the patient had little or noresidual tumor sink for the circulating antibodies, the levels detectedat Week 5 were already much higher than in the patient who had amoderate tumor mass.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

All of the patents and publications referred to herein are incorporatedby reference in their entirety.

For purposes of interpreting this specification, the followingdefinitions will generally apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa. In the eventthat any definition set forth conflicts with any document incorporatedherein by reference, the definition set forth below shall prevail.

“Purinergic receptor” generally refers to a receptor that uses a purine(such as ATP) as a ligand.

“P2X₇ receptor” generally refers to a purinergic receptor formed fromthree protein subunits or monomers, with at least one of the monomershaving an amino acid sequence substantially as shown in SEQ ID No:1. Tothe extent that P2X₇ receptor is formed from three monomers, it is a“trimer” or “trimeric”. “P2X₇ receptor” may be a functional ornon-functional receptor as described below. “P2X₇ receptor” encompassesnaturally occurring variants of P2X₇ receptor, e.g., wherein the P2X₇monomers are splice variants, allelic variants and isoforms includingnaturally-occurring truncated or secreted forms of the monomers formingthe P2X₇ receptor (e.g., a form consisting of the extracellular domainsequence or truncated form of it), naturally-occurring variant forms(e.g., alternatively spliced forms) and naturally-occurring allelicvariants. In certain embodiments of the invention, the native sequenceP2X₇ monomeric polypeptides disclosed herein are mature or full-lengthnative sequence polypeptides comprising the full-length amino acidssequence shown in SEQ ID No:1. In certain embodiments the P2X₇ receptormay have an amino acid sequence that is modified, for example various ofthe amino acids in the sequence shown in SEQ ID No:1 may be substituted,deleted, or a residue may be inserted.

“Functional P2X₇ receptor” generally refers to a form of the P2X₇receptor having a binding site or cleft for binding to ATP. When boundto ATP, the receptor forms non-selective sodium/calcium channel thatconverts to a pore-like structure that enables the ingress of calciumions into the cytosol, one consequence of which may be programmed celldeath. In normal homeostasis, expression of functional P2X₇ receptors isgenerally limited to cells that undergo programmed cell death such asthymocytes, dendritic cells, lymphocytes, macrophages and monocytes.There may also be some expression of functional P2X₇ receptors onerythrocytes and other cell types.

“Non-functional P2X₇ receptor” generally refers to a form of a P2X₇receptor having a conformation whereby the receptor is unable to form anapoptotic pore, but which is still able to operate as a non-selectivechannel through the maintenance of a single functional ATP binding sitelocated between adjacent monomers. One example arises where one or moreof the monomers has a cis isomerisation at Pro210 (according to SEQ IDNo:1). The isomerisation may arise from any molecular event that leadsto misfolding of the monomer, including for example, mutation of monomerprimary sequence or abnormal post translational processing. Oneconsequence of the isomerisation is that the receptor is unable to bindto ATP at one or two ATP binding sites on the trimer and as aconsequence not be able to extend the opening of the channel. In thecircumstances, the receptor cannot form a pore and this limits theextent to which calcium ions may enter the cytosol. Non-functional P2X₇receptors are expressed on a wide range of epithelial and haematopoieticcancers.

“Cancer associated—P2X₇ receptors” are generally P2X₇ receptors that arefound on cancer cells (including, pre-neoplastic, neoplastic, malignant,benign or metastatic cells), but not on non cancer or normal cells.

“E200 epitope” generally refers to an epitope exposed on anon-functional P2X₇ receptor. In humans the sequence isGHNYTTRNILPGLNITC (SEQ ID NO: 2).

“E300 epitope” generally refers to an epitope exposed on anon-functional P2X₇ receptor. In humans the sequence isKYYKENNVEKRTLIKVF (SEQ ID NO: 3).

“Composite epitope” generally refers to an epitope that is formed fromthe juxtaposition of the E200 and E300 epitopes or parts of theseepitopes.

“Antibodies” or “immunoglobulins” or “Igs” are gamma globulin proteinsthat are found in blood, or other bodily fluids of verterbrates thatfunction in the immune system to bind antigen, hence identifying and/orneutralizing foreign objects.

Antibodies are generally a heterotetrameric glycoprotein composed of twoidentical light (L) chains and two identical heavy (H) chains. Each Lchain is linked to a H chain by one covalent disulfide bond. The two Hchains are linked to each other by one or more disulfide bonds dependingon the H chain isotype. Each H and L chain also has regularly spacedintrachain disulfide bridges.

H and L chains define specific Ig domains. More particularly, each Hchain has at the N-terminus, a variable domain (V_(H)) followed by threeconstant domains (C_(H)) for each of the α and γ chains and four C_(H)domains for μ and ε isotypes. Each L chain has at the N-terminus, avariable domain (V_(L)) followed by a constant domain (CO at its otherend. The V_(L) is aligned with the V_(H) and the C_(L) is aligned withthe first constant domain of the heavy chain (C_(H)1).

Antibodies can be assigned to different classes or isotypes. There arefive classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Lchain from any vertebrate species can be assigned to one of two clearlydistinct types, called kappa and lambda, based on the amino acidsequences of their constant domains.

The constant domain includes the Fc portion which comprises thecarboxy-terminal portions of both H chains held together by disulfides.The effector functions of antibodies such as ADCC are determined bysequences in the Fc region, which region is also the part recognized byFc receptors (FcR) found on certain types of cells.

The pairing of a V_(H) and V_(L) together forms a “variable region” or“variable domain” including the amino-terminal domains of the heavy orlight chain of the antibody. The variable domain of the heavy chain maybe referred to as “V_(H).” The variable domain of the light chain may bereferred to as “V_(L).” The V domain contains an “antigen binding site”which affects antigen binding and defines specificity of a particularantibody for its particular antigen. V regions span about 110 amino acidresidues and consist of relatively invariant stretches called frameworkregions (FRs) (generally about 4) of 15-30 amino acids separated byshorter regions of extreme variability called “hypervariable regions”(generally about 3) that are each generally 9-12 amino acids long. TheFRs largely adopt a 13-sheet configuration and the hypervariable regionsform loops connecting, and in some cases forming part of, the 13-sheetstructure.

“Hypervariable region” refers to the regions of an antibody variabledomain which are hypervariable in sequence and/or form structurallydefined loops. Generally, antibodies comprise six hypervariable regions;three in the V_(H) (H1, H2, H3), and three in the V_(L) (L1, L2, L3).

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues herein defined.

An “antigen binding site” generally refers to a molecule that includesat least the hypervariable and framework regions that are required forimparting antigen binding function to a V domain. An antigen bindingsite may be in the form of an antibody or an antibody fragment, (such asa dAb, Fab, Fd, Fv, F(ab′)₂ or scFv) in a method described herein.

An “intact” or “whole” antibody is one which comprises anantigen-binding site as well as a C_(L) and at least heavy chainconstant domains, C_(H)I, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof.

“whole antibody fragments including a variable domain” include Fab,Fab′, F(ab)₂, and Fv fragments; diabodies; linear antibodies,single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

The “Fab fragment” consists of an entire L chain along with the variableregion domain of the H chain (V_(H)), and the first constant domain ofone heavy chain (C_(H)I). Each Fab fragment is monovalent with respectto antigen binding, i.e., it has a single antigen-binding site.

A “Fab′ fragment” differs from Fab fragments by having additional fewresidues at the carboxy terminus of the C_(H)I domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group.

A “F(ab)₂ fragment” roughly corresponds to two disulfide linked Fabfragments having divalent antigen-binding activity and is still capableof cross-linking antigen.

An “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and binding site. This fragment consists of a dimerof one heavy and one light chain variable region domain in tight,non-covalent association.

In a single-chain Fv (scFv) species, one heavy and one light chainvariable domain can be covalently linked by a flexible peptide linkersuch that the light and heavy chains can associate in a “dimeric”structure analogous to that in a two-chain Fv species. From the foldingof these two domains emanate six hypervariable loops (3 loops each fromthe H and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedto form a single polypeptide chain. Preferably, the scFv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the scFv to form the desired structure for antigenbinding.

A “single variable domain” is half of an Fv (comprising only three CDRsspecific for an antigen) that has the ability to recognize and bindantigen, although at a lower affinity than the entire binding site

“Diabodies” refers to antibody fragments with two antigen-binding sites,which fragments comprise a heavy-chain variable domain (V_(H)) connectedto a light-chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L)). The small antibody fragments are prepared by constructingsFv fragments (see preceding paragraph) with short linkers (about 5-10residues) between the V_(H) and V_(L) domains such that interchain butnot intra-chain pairing of the V domains is achieved, resulting in abivalent fragment, i.e., fragment having two antigen-binding sites.

Diabodies may be bivalent or bispecific. Bispecific diabodies areheterodimers of two “crossover” sFv fragments in which the V_(H) andV_(L) domains of the two antibodies are present on different polypeptidechains. Triabodies and tetrabodies are also generally known in the art.

An “isolated antibody” is one that has been identified and separatedand/or recovered from a component of its pre-existing environment.Contaminant components are materials that would interfere withtherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes.

A “human antibody” refers to an antibody that possesses an amino acidsequence that corresponds to that of an antibody produced by a human.Human antibodies can be produced using various techniques known in theart, including phage-display libraries. Human antibodies can be preparedby administering the antigen to a transgenic animal that has beenmodified to produce such antibodies in response to antigenic challenge,but whose endogenous loci have been disabled.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts.

Monoclonal antibodies are highly specific, being directed against asingle antigenic site or determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. Monoclonalantibodies may be prepared by the hybridoma methodology. The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques.

The term “anti-P2X₇ receptor antibody” or “an antibody that binds toP2X₇ receptor” refers to an antibody that is capable of binding P2X₇receptor with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting P2X₇ receptor,typically non-functional P2X₇ receptor. Preferably, the extent ofbinding of an P2X₇ receptor antibody to an unrelated protein is lessthan about 10% of the binding of the antibody to P2X₇ receptor asmeasured, e.g., by a radioimmunoassay (RIA), Enzyme-Linked ImmunosorbentAssay (ELISA), Biacore or Flow Cytometry. In certain embodiments, anantibody that binds to P2X₇ receptor has a dissociation constant (Kd) of<1 □M, <100 nM, <10 nM, <1 nM, or <0.1 nM. An anti non-functional P2X₇receptor antibody is generally one having some or all of theseserological characteristics and that binds to non-functional P2X₇receptors but not to functional P2X₇ receptors.

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable region thereof that result in an improvementin the affinity of the antibody for antigen, compared to a parentantibody that does not possess those alteration(s). Preferred affinitymatured antibodies will have nanomolar or even picomolar affinities forthe target antigen. Affinity matured antibodies are produced byprocedures known in the art.

A “blocking” antibody” or an “antagonist” antibody is one that inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody, which mimics atleast one of the functional activities of a polypeptide of interest.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity, which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention.

“Epitope” generally refers to that part of an antigen that is bound bythe antigen binding site of an antibody. An epitope may be “linear” inthe sense that the hypervariable loops of the antibody CDRs that formthe antigen binding site bind to a sequence of amino acids as in aprimary protein structure. In certain embodiments, the epitope is a“conformational epitope” i.e. one in which the hypervariable loops ofthe CDRs bind to residues as they are presented in the tertiary orquaternary protein structure.

‘Treatment’ generally refers to both therapeutic treatment andprophylactic or preventative measures.

Subjects requiring treatment include those already having a benign,pre-cancerous, or non-metastatic tumor as well as those in which theoccurrence or recurrence of cancer is to be prevented.

The objective or outcome of treatment may be to reduce the number ofcancer cells; reduce the primary tumor size; inhibit (i.e., slow to someextent and preferably stop) cancer cell infiltration into peripheralorgans; inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the disorder.

Efficacy of treatment can be measured by assessing the duration ofsurvival, time to disease progression, the response rates (RR), durationof response, and/or quality of Iife.

In one embodiment, the method is particularly useful for delayingdisease progression.

In one embodiment, the method is particularly useful for extendingsurvival of the human, including overall survival as well as progressionfree survival.

In one embodiment, the method is particularly useful for providing acomplete response to therapy whereby all signs of cancer in response totreatment have disappeared. This does not always mean the cancer hasbeen cured.

In one embodiment, the method is particularly useful for providing apartial response to therapy whereby there has been a decrease in thesize of one or more tumors or lesions, or in the extent of cancer in thebody, in response to treatment.

“Pre-cancerous” or “pre-neoplasia” generally refers to a condition or agrowth that typically precedes or develops into a cancer. A“pre-cancerous” growth may have cells that are characterized by abnormalcell cycle regulation, proliferation, or differentiation, which can bedetermined by markers of cell cycle.

In one embodiment, the cancer is pre-cancerous or pre-neoplastic.

In one embodiment, the cancer is a secondary cancer or metastases. Thesecondary cancer may be located in any organ or tissue, and particularlythose organs or tissues having relatively higher hemodynamic pressures,such as lung, liver, kidney, pancreas, bowel and brain.

Other examples of cancer include blastoma (including medulloblastoma andretinoblastoma), sarcoma (including liposarcoma and synovial cellsarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma,and islet cell cancer), mesothelioma, schwannoma (including acousticneuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoidmalignancies, lung cancer including small-cell lung cancer (SGLG),non-small cell lung cancer (NSGLG), adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer (including metastaticbreast cancer), colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophagael cancer, tumors of the biliary tract, as well as head and neckcancer.

“A condition or symptom associated” [with the cancer] may be anypathology that arises as a consequence of, preceding, or proceeding fromthe cancer. For example, where the cancer is a skin cancer, thecondition or relevant symptom may be microbial infection. Where thecancer is a secondary tumor, the condition or symptom may relate toorgan dysfunction of the relevant organ having tumor metastases. In oneembodiment, the methods of treatment described herein are for theminimisation or treatment of a condition or symptom in an individualthat is associated with a cancer in the individual.

A “non self” molecule, such as a “non self” antigen binding site, or“non self” antibody generally refers to a molecule that has beenproduced outside of, or exogenous to, a body in which the molecule is tobe provided, for example, for treatment. As an example, synthetic orrecombinant molecules are “non self”. Further, a molecule that isgenerated in one individual and administered to another individual fortreatment is “non self”. “Non self” antigen binding sites and antibodiesmay be used in accordance with the invention for adoptive transfer ofimmunity, for example, as occurs in antibody infusion. In contrast, amolecule that is generated inside an individual that is to be treatedwith that molecule, is generally a “self” or “endogenous” molecule. Oneexample of a “self” molecule is an antigen binding site or antibody thatis generated, or arises from an adaptive immune response to immunogen.

“level of non self antigen binding sites in circulation” in theindividual generally refers to the concentration of antigen binding sitein a body fluid, preferably peripheral blood.

A “substantially undetectable level of non self antigen binding sites incirculation” generally refers to a concentration of exogenous antigenbinding sites (i.e. those that have been administered by adoptivetransfer) that is at least half of the concentration of the antigenbinding sites in circulation at the time of administration of theantigen binding sites, preferably 25%, or 10%, or 5% or 1% of saidconcentration, or otherwise less than 0.001 mg/kg of the individual. Thephrase may also refer to a circumstance where antigen binding sites thathave been given for the purpose of cancer immunotherapy cannot bedetected at all.

A cancer that is “substantially undetectable” generally refers to acircumstance where therapy has depleted the size, volume or otherphysical measure of a cancer so that using relevant standard detectiontechniques such as in vivo imaging, the cancer, as a consequence of thetherapy, is not clearly detectable. The phrase also refers to thecircumstance where the cancer cannot be detected at all.

“forming an immune response” generally refers to invoking or inducingantigen specific immunity via the adaptive immune system. As isgenerally understood in the art, induction of antigen specific immunityis distinguished from adoptive transfer of immunity, standard cancerimmunotherapy by administration of exogenous or non self antibody beingone example of the latter.

Individuals Selected for Treatment

Generally, the individuals selected for treatment according to a methoddescribed above are those who have received, or who are continuing toreceive antibody immunotherapy, for treatment of cancer. Antibodyimmunotherapy generally means the administration of exogenous,(otherwise known as or “non self”) antibodies to an individual requiringtreatment, as in the case of adoptive transfer of antibody. For example,the individual may have received any one of the therapeutic antibodiesthat have received regulatory approval for indications related tooncology. Avastin, Herceptin, Rituxan are examples. Typically theindividual has received or is continuing to receive an anti P2X₇receptor antibody.

In one embodiment, the individual may have received immunotherapyleading to undetectable tumour mass and no longer has detectablecirculating exogenous antibody at the time of immunisation.

Further, the individual selected for treatment according to a methoddescribed above may or may not have detectable cancer at the time oftreatment. Where the individual does not have detectable cancer, aprimary or secondary humoral response is more easily detected because,with the cancer being present in substantially undetected amount, thereis very little non-functional P2X₇ receptor available to remove IgM orIgG from body fluid. This point is demonstrated in FIGS. 1 and 2.

The purpose of the treatment according to the above described methods isto at least minimise the progression of cancer by induction or formationof an immune response in the individual to a non-functional P2X₇receptor. Therefore, the individual selected for treatment must becapable of generating an immune response sufficient for meeting thispurpose. Generally the desired immune response includes a capacity toproduce either or both of circulating IgM and IgG when the individual ischallenged by cancer, as in recurrence of cancer. In one embodiment, thepresence of an immune response to non-functional P2X₇ receptor ismeasured or assessed when the individual does not have detectablecancer. In these circumstances it is believed that the absence of ananti-P2X₇ receptor antibody-absorbing mass in the form of tumourincreases the likelihood of there being a higher systemic titre ofanti-P2X₇ receptor antibody.

Individuals having a capacity to generate the immune response describedherein may be selected or screened by a variety of methods well known inthe art for detection of immunodeficiency. Typically, the individualselected for treatment will be one having at least one white blood cellcomponent count within normal parameters. For example, an individual forinclusion is generally one having a white blood cell count of between4.0 to 11.0×10⁹/L, or a lymphocyte count of between 1.0 to 4.4×10⁹/L.Neutrophil count may be between 1.9-7.8×10⁹/L; monocyte count between0.2-1.0×10⁹/L, eosinophil less than about 5.0×10⁹/L and basophil lessthan about 0.2×10⁹/L.

It will be understood that in certain embodiments the cell count for anyone of these blood cell components may fall outside these stated ranges,particularly in circumstances where the individual has a form of bloodcancer, for example CML, CLL etc.

Generally an important factor is the lymphocyte count and/or monocytecount. In more detail, where either or both of these counts aresignificantly below the stated ranges for these components, theindividual may be less likely to respond to administration of theimmunogen.

In another embodiment, the selection of an individual for therapy mayinvolve a screening step for screening whether the individual ishomozygous for non-functional P2X₇ receptor expression. In more detail,a small percentage of the Caucasian population is understood to havenon-functional P2X₇ receptor expression on non cancer cells such ascertain thymocytes, dendritic cells, lymphocytes, macrophages, monocytesand erythrocytes. Other individuals may be heterozygotic for thisexpression. In one embodiment, individuals who are homozygotic fornon-functional P2X₇ receptor expression are screened for and excludedfrom treatment, in which case individuals included for treatment do notexpress non-functional P2X₇ receptor on non cancer cells, or otherwisehave heterozygotic expression.

Where the individual is continuing to receive antibody immunotherapy, inone embodiment the antibody immunotherapy is allowed to continue to thedesired clinical endpoint. Typically the desired clinical endpoint is areduction of cancer to substantially undetectable levels. During, or atthe completion of immunotherapy, the capacity of the individual to form,or generate an immune response to a P2X₇ receptor is then assessed.Where the assessment reveals that the individual is likely to benefitfrom immunisation with P2X₇ immunogen, the individual is thenadministered with immunogen.

In a preferred form of the invention, the level of non self or exogenousantigen binding sites in circulation in the individual arising fromantibody immunotherapy is substantially undetectable at the time thatthe immune response is formed in the individual. Importantly, where thenon self antigen binding site binds to non functional P2X₇ receptor orcancer-associated P2X₇ receptor, a key finding of the inventor is thatefficacy of antibody treatment, particularly when cancer cells are invery low copy number, or otherwise substantially undetectable, decreasesat higher circulating concentrations of antigen binding sites. This isbelieved to be a function of the low copy number of P2X₇ receptors oncancer cells relative to the high concentration of antigen binding sitesthat arise in standard antibody immunotherapy. Specifically, in theExamples herein, the inventor has found that as the circulatory level ofantigen specific binding sites increase, and the number of cancer cellsdecrease, there is a much higher likelihood of crowding of the P2X₇receptor by antigen binding sites that block antigen specific binding ofthe receptor. This blockage increases the likelihood that the intendedcytotoxic, apoptotic or other effects of antigen specific binding by anantigen binding site will not be possible. One can determine the levelof exogenous antigen binding sites in circulation by any standardserological technique capable of detecting antibody in fluid, onepreferred example being ELISA using an antibody to capture antigenbinding sites.

Further to the above, while not wanting to be bound by hypothesis, theinventors consider that administration of immunisation at a time whereinfded antibody is present increases the risk that the infused antibodycould bind to the immunogen, resulting in immune complex formation andclearance, thereby avoiding antigen presentation and induction ofantigen specific immunity. Therefore, in certain embodiments it isparticularly useful to wait until the level of non self or exogenousantigen binding sites have been cleared from circulation beforeinduction of the antigen specific immune response to immunogen.

In embodiments where the infused antibody cannot bind the immunogen(i.e. because the antibody is not specific for the immunogen, forexample where the antibody binds to a biomarker that is unrelated to theimmunogen) the immunisation with immunogen may occur when there isdetectable infused antibody, or before antibody infusion occurs.

Antigen Binding Sites and Administration

An individual to be treated in accordance with the methods describedherein may be one who has received, or is to receive any one of thetherapeutic antibodies indicated for oncology. Preferably, theindividual has received or is continuing to receive an anti P2X₇receptor antibody.

Typically the antigen binding site is one that discriminates betweenfunctional and non-functional P2X₇ receptors, so as to bind tonon-functional receptors, but not to functional receptors. Examples ofthese antigen binding sites are those that bind to the E200 epitope,E300 epitope or composite epitope as for example in PCT/AU2002/000061,PCT/AU2002/001204, PCT/AU2007/001540, PCT/AU2007/001541,PCT/AU2008/001364, PCT/AU2008/001365, PCT/AU2009/000869 andPCT/AU2010/001070, all of which are incorporated by reference.

Regardless of specificity, (i.e. P2X₇ receptor specific or otherwise),the antigen binding site may take the form of a whole antibody, or awhole antibody fragment such as a Fab, a Fab′, a F(ab′)₂, and Fv, asingle chain Fv, or a single variable domain.

The antigen binding site may be syngeneic, allogeneic or xenogeneic.

Typically the antigen binding site is non self or exogenous meaning thatit has been found or isolated outside of the individual who is treatedaccording to the methods of the invention.

The antigen binding site may be affinity matured.

The antigen binding site may have multiple specificities or valencies.

The antigen binding site may be adapted so as to be suited toadministration by a selected method.

The antibody may be a whole antibody of any isotype. The antibody may beone obtained from monoclonal or polyclonal antisera. The antibody may beproduced by hybridoma, or by recombinant expression, or may be obtainedfrom serum for example as obtainable from a mammal, particularly a humanor mouse. The antibody may also be obtained from an avian.

The antibody may be chimeric, i.e. one containing human variable domainsand non human constant domains. Alternatively, it may be humanized, i.eone formed by grafting non human CDRs onto a human antibody framework.Still further, the antibody may be fully human.

The antibody may be modified with respect to effector function, so as toenhance, e.g., the effectiveness of the antibody in treating cancer.

Where the antibody is an antibody fragment, the antibody fragment isselected from the group consisting of a dAb, Fab, Fd, Fv, F(ab′)₂, scFvand CDR.

Dosage amount, dosage frequency, routes of administration etc aredescribed in detail below.

Methods of preparing and administering antibodies to a subject in needthereof are well known to, or are readily determined by those skilled inthe art. The route of administration may be, for example, oral,parenteral (e.g. intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, intradermal, rectal or vaginal), byinhalation or topical. One form for administration would be a solutionfor injection, in particular for intravenous or intraarterial injectionor drip, comprising a buffer (e.g. acetate, phosphate or citratebuffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent(e.g. human albumin). In other methods antibodies can be delivereddirectly to the site of disease thereby increasing the exposure of thediseased cell or tissue to the antibody.

Preparations for parenteral administration includes sterile aqueous(aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media) or non-aqueous(non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate) solutions, suspensions, and emulsions. Pharmaceuticallyacceptable carriers include 0.01-0.1M and preferably 0.05M phosphatebuffer or 0.9% saline. Other common parenteral vehicles include sodiumphosphate solutions, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers, such as those based onRinger's dextrose, and the like. Preservatives and other additives mayalso be present such as for example, antimicrobials, antioxidants,chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions, in such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.,16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., antigen binding site) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle, which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying, freeze-drying and spray drying, which yields a powder ofan active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The preparations forinjections are processed, filled into containers such as ampoules, bags,bottles, syringes or vials, and sealed under aseptic conditionsaccording to methods known in the art. Further, the preparations may bepackaged and sold in the form of a kit. Such articles of manufacturewill preferably have labels or package inserts indicating that theassociated compositions are useful for treating a subject sufferingfrom, or predisposed to disorders.

Effective doses of the compositions of the present invention, fortreatment of disorders, as described herein, vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Treatment dosages may be titrated usingroutine methods known to those of skill in the art to optimize safetyand efficacy.

For treatment of certain disorders with an antibody, the dosage canrange, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2mg/kg, etc.), of the host body weight. For example dosages can be 1mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the invention.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moreantigen binding sites with different binding specificities areadministered simultaneously, in which case the dosage of each antigenbinding site administered falls within the ranges indicated.

The antibody for binding to a non-functional P2X₇ receptor expressed ona cell can be administered on multiple occasions. Intervals betweensingle dosages can be weekly, monthly or yearly. Intervals can also beirregular as indicated by measuring blood levels of target polypeptideor target molecule in the patient. In some methods, dosage is adjustedto achieve a plasma polypeptide concentration of 1-1000 ug/mL and insome methods 25-300 ug/mL. Alternatively, the antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. The half-life of anantibody can also be prolonged via fusion to a stable polypeptide ormoiety, e.g., albumin or PEG. In general, humanized antibodies show thelongest half-life, followed by chimeric antibodies and nonhumanantibodies. In one embodiment, the antibody can be administered inunconjugated form. In another embodiment the antibody can beadministered multiple times in conjugated form. In certain therapeuticapplications, a relatively high dosage (e.g., up to 400 mg/kg of antiP2X₇ binding molecule, e.g., antibody per dose), at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. The amounts can be severallogs lower (i.e. 2 to 3 logs lower) where the antibody is conjugated toa radioisotope or cytotoxic drug.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment, in some methods, agents are injecteddirectly into a particular tissue where non-functional P2X₇ receptorcells have accumulated, for example intracranial injection.Intramuscular injection or intravenous infusion are preferred foradministration of antibody.

An antibody can optionally be administered in combination with otheragents that are effective in treating the disorder or condition in needof treatment (e.g., prophylactic or therapeutic). Examples are agentscommonly used for chemotherapy or radiotherapy in oncology. Additionallyor alternatively, the antibody or agent may be administered before,during or after surgical intervention for resection or removal of tumoror tissue.

Immunogens and Forming an Immune Response

The methods of the invention described herein require the formation ofan immune response in an individual to be treated to a P2X₇ receptor,especially a non-functional P2X₇ receptor. Generally the immunogen,which is used for the purpose, is one which elicits an immune responseto non-functional P2X₇ but not to functional P2X₇ receptors.

The immunogen may include or consist of a peptide including a sequenceof a P2X₇ receptor. The peptide may contain at least one sequence thatis capable of being presented on a major histocompatibility complexclass II molecule or, that is capable of interacting with a B-cellreceptor or a B-cell membrane bound-immunoglobulin. Typically thepeptide includes a sequence of a human P2X₇ receptor or fragmentthereof.

A range of peptide immunogens are known and discussed inPCT/AU2002/000061, PCT/AU2002/000061, PCT/AU2008/001364 andPCT/AU2009/000869, the contents of which are incorporated in entirety.

Exemplary peptides immunogens within these specifications, which includeepitopes for generating an immune response to a non-functional P2X₇receptor are described below.

PCT application Peptide immunogen sequence PCT/AU2002/000061,GHNYTTRNILPGLNITC PCT/AU2002/000061 (SEQ ID NO: 2) PCT/AU2008/001364KYYKENNVEKRTLIKVF (SEQ ID NO: 3) PCT/AU2009/000869GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 4)

It will be understood that these are merely examples of possibleimmunogens useful for forming an immune response according to themethods of the invention described herein. Further, the inventionincludes the use of other peptides as described in these applicationsuseful for forming an immune response to non-functional P2X₇ receptors.

Typically the immunisation regime involves 2 or more immunisations. In afirst immunisation, the objective may be to develop an IgM response toimmunisation. A second immunisation may be to develop an IgG response.Further immunisations may be to boost the IgG response, as discussedfurther below.

Where the immunogen is a peptide, the peptide may be provided in anamount of about 0.1 to 1 mg per administration, preferably about 0.25 to0.75 mg, preferably about 0.5 mg.

A further administration of about 0.3 mg peptide may be applied as aboost.

In one embodiment, a first immunisation is performed when thecirculating level of antigen binding sites that had been administeredfor antibody immunotherapy is substantially undetectable. In otherwords, circulating antibody to the relevant cancer biomarker cannot bedetected in peripheral blood. The level of IgM production is thenmonitored over the following weeks. At about 4 to 5 weeks after firstimmunisation, the level of IgM antibody is likely to have decreased tonegligible circulating levels. At this point, a second immunisation isthen performed and the level of IgG production is monitored over thefollowing weeks.

After boosting, the level of antibody produced may be 0.1-25 mg/kg, forexample from 0.1 to 10 mg/kg, preferably 5 mg/kg, or from 10 to 25mg/kg, preferably 15 mg/kg and more than 10 mg/kg. Whether this amountis detected in circulation will depend on whether there is existingtumour mass. Where there is existing tumour mass capable of binding toantibody formed by the humoral response, the level of antibody detectedin circulation may be at the lower end of this range, or indeed outsidethe lower end of the range (i.e. less than 0.1 mg/kg), or otherwisesubstantially undetectable. Where there is no detectable tumour mass,the level of antibody formed from the humoral response may be at thehigher end of this range, although in certain embodiments, in thesecircumstances, an amount of about 5 mg/kg antibody may be sufficient.Further testing of immunity over the following months/years may beperformed and boosting immunisations may be provided as required.

The degree or number of boosts may depend on the patient status andresponse. Where scans or lack of free circulating antibody areindicative of extant tumour burden, then boosts may be performedmonthly, ideally to ensure sufficient reaction of the immune system.Where the free levels of antibody in serum rise, the boosts may then beeased off and perhaps applied 6-12 monthly subject to clinicalobservation.

As discussed above, the immune response may target a biomarker that isdifferent to the biomarker that has been targeted by antibodyimmunotherapy. For example, anti CD20 antibody may be used for antibodyimmunotherapy and a non-functional P2X₇ immunogen used for generating animmune response.

In another embodiment, a single biomarker is targeted by antibodyimmunotherapy and immunisation. For example, a monoclonal antibodydirected to one epitope on a P2X₇ receptor (such as the E300 epitope)may be used for antibody immunotherapy, and an immunogen for forming animmune response that targets a different epitope (such as the E200epitope) on P2X₇ may be used for immunisation.

A peptide immunogen for use in a method of the invention herein may havea length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or 26 residues.

In one embodiment, the immunogen for forming an immune responseaccording to a method of the invention is a peptide having a sequence ofa P2X₇ receptor that may or may not have Pro210 in cis conformation.

The immunogen may be in the form of a P2X₇ extracellular domain or anyone or more of the P2X₇ isoforms. The immunogen may be provided foradministration in a soluble form or associated with a solid phase suchas a cell membrane, bead, or other surface.

Methods for screening peptides that can be used as an immunogen to forman immune response according to the methods of the invention herein aredisclosed herein. One example includes the use of erythrocytes in arosetting assay. In this assay an antibody that binds to functionalreceptors is used as a positive control in which rossettes are observed.A test antibody is determined not to bind to functional receptors if itfails to form rossettes. It is determined to bind to non-functionalreceptors if it is observed to bind to a non-functionalreceptor-expressing cell line, including those discussed herein.

The peptides of the invention can be made by any number of techniquesknown in the art including solid phase synthesis and recombinant DNAtechnology.

As is known in the art, a carrier is a substance that may be conjugatedto a peptide epitope thereby enhancing immunogenicity. Some carriers dothis by binding to multiple peptides so as to provide an antigen ofincreased molecular weight to the host in which the immune response isto be developed.

Preferred carriers include bacterial toxins or toxoids. Other suitablecarriers include the N. meningitides outer membrane protein, albuminsuch as bovine serum albumin, synthetic peptides, heat shock proteins,KLH, Pertussis proteins, protein D from H. influenza and toxin A, B or Cfrom C. difficile.

When the carrier is a bacterial toxin or toxoid, diphtheria or tetanustoxoids are preferred.

Preferably the carrier contains functional groups that can react withthe peptide of the invention, or may be modified to be capable ofreacting with the peptide.

The immunogen may be administered subcutaneously, intradermally and/orintramuscularly.

Adjuvants

In a preferred form, the composition for forming an immune response to aP2X₇ receptor for use in the methods of the invention described hereinincludes an adjuvant or compound for potentiating an immune response.

A large number of adjuvants are known; See also Allison (1998, Dev.Biol. Stand., 92:3-11; incorporated herein by reference), Unkeless etal. (1998, Annu. Rev. Immunol., 6:251-281), and Phillips et al. (1992,Vaccine, 10:151-158). Exemplary adjuvants that can be utilized inaccordance with the invention include, but are not limited to,cytokines, aluminium salts (e.g., aluminium hydroxide, aluminiumphosphate, etc.; Baylor et al., Vaccine, 20:S18, 2002), gel-typeadjuvants (e.g., calcium phosphate, etc.); microbial adjuvants (e.g.,immunomodulatory DNA sequences that include CpG motifs; endotoxins suchas monophosphoryl lipid A (Ribi et al., 1986, Immunology andImmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY,p407, 1986); exotoxins such as cholera toxin, E. coli heat labile toxin,and pertussis toxin; muramyl dipeptide, etc.); oil-emulsion andemulsifier-based adjuvants (e.g., Freund's Adjuvant, MF59 [Novartis],SAF, etc.); particulate adjuvants (e.g., liposomes, biodegradablemicrospheres, etc.); synthetic adjuvants (e.g., nonionic blockcopolymers, muramyl peptide analogues, polyphosphazene, syntheticpolynucleotides, etc.); and/or combinations thereof. Other exemplaryadjuvants include some polymers (e.g., polyphosphazenes; described inU.S. Pat. No. 5,500,161), Q57, saponins (e.g., QS21, Ghochikyan et al.,Vaccine, 24:2275, 2006), squalene, tetrachlorodecaoxide, CPG 7909(Cooper et al., Vaccine, 22:3136, 2004),poly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., Vaccine,16:92, 1998), interferon-γ (Cao et al., Vaccine, 10:238, 1992), blockcopolymer P1205 (CRL1005; Katz et al., Vaccine, 18:2177, 2000),interleukin-2 (IL-2; Mbwuike et al., Vaccine, 8:347, 1990), polymethylmethacrylate (PMMA; Kreuter et al., J. Pharm. ScL, 70:367, 1981), etc.

In one embodiment, a peptide immunogen containing a sequence of a P2X₇receptor is provided on the surface of a bacteriophage for immunisationof an individual according to a method of the invention describedherein.

Cancers and Conditions Associated Therewith

Pre-neoplastic, neoplastic and metastatic diseases are particularexamples to which the methods of the invention may be applied. Broadexamples include breast tumors, colorectal tumors, adenocarcinomas,mesothelioma, bladder tumors, prostate tumors, germ cell tumor,hepatoma/cholongio, carcinoma, neuroendocrine tumors, pituitaryneoplasm, small round cell tumor, squamous cell cancer, melanoma,atypical fibroxanthoma, seminomas, nonseminomas, stromal leydig celltumors, Sertoli cell tumors, skin tumors, kidney tumors, testiculartumors, brain tumors, ovarian tumors, stomach tumors, oral tumors,bladder tumors, bone tumors, cervical tumors, esophageal tumors,laryngeal tumors, liver tumors, lung tumors, vaginal tumors and Wilm'stumor.

Examples of particular cancers include but are not limited toadenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDSrelated cancers, acoustic neuroma, acute lymphocytic leukemia, acutemyeloid leukemia, adenocystic carcinoma, adrenocortical cancer,agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma,ameloblastoma, angiokeratoma, angiolymphoid hyperplasia witheosinophilia, angioma sclerosing, angiomatosis, apudoma, anal cancer,angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia,basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer,brain stem glioma, brain and CNS tumors, breast cancer, branchioma, CNStumors, carcinoid tumors, cervical cancer, childhood brain tumors,childhood cancer, childhood leukemia, childhood soft tissue sarcoma,chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronicmyeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma,carcinoma (e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal,Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oatcell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell,and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcomaphyllodies, cementoma, chordoma, choristoma, chondrosarcoma,chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma,cylindroma, cystadenocarcinoma, cystadenoma,dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor,ductal carcinoma, dysgerminoam, endocrine cancers, endometrial cancer,ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile ductcancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tubecancer, fanconi anaemia, fibroma, fibrosarcoma, gall bladder cancer,gastric cancer, gastrointestinal cancers,gastrointestinal-carcinoid-tumor, genitourinary cancers, germ celltumors, gestationaltrophoblastic-disease, glioma, gynaecologicalcancers, giant cell tumors, ganglioneuroma, glioma, glomangioma,granulosa cell tumor, gynandroblastoma, haematological malignancies,hairy cell leukemia, head and neck cancer, hepatocellular cancer,hereditary breast cancer, histiocytosis, Hodgkin's disease, humanpapillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer,hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma,hemangiosarcoma, hemangiosarcoma, histiocytic disorders, histiocytosismalignant, histiocytoma, hepatoma, hidradenoma, hondrosarcoma,immunoproliferative small, opoma, ontraocular melanoma, islet cellcancer, Kaposi's sarcoma, kidney cancer, langerhan's cell-histiocytosis,laryngeal cancer, leiomyosarcoma, leukemia, li-fraumeni syndrome, lipcancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, leigomyosarcoma, leukemia(e.g. b-cell, mixed cell, null-cell, t-cell, t-cell chronic,htiv-ii-associated, lymphangiosarcoma, lymphocytic acute, lymphocyticchronic, mast-cell and myeloid), leukosarcoma, leydig cell tumor,liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma,lymphagioma, lymphagiomyoma, lymphangiosarcoma, male breast cancer,malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, Merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,myeloma, myeloproliferative disorders, malignant carcinoid syndromecarcinoid heart disease, medulloblastoma, meningioma, melanoma,mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma,myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer,nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakagesyndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(nscic),neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis,neurofibroma, neuroma, neoplasms (e.g. bone, breast, digestive system,colorectal, liver), ocular cancers, oesophageal cancer, oral cavitycancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreascancer, paranasal cancer, parathyroid cancer, parotid gland cancer,penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer,polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovariancarcinoma, papilloma, paraganglioma, paraganglioma nonchromaffin,pinealoma, plasmacytoma, protooncogene,rare-cancers-and-associated-disorders, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome,reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma,schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (scic),small intestine cancer, soft tissue sarcoma, spinal cord tumors,squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,sarcoma (e.g. Ewing's experimental, Kaposi's and mast-cell sarcomas),Sertoli cell tumor, synovioma, testicular cancer, thymus cancer, thyroidcancer, transitional-cell-cancer-(bladder),transitional-cell-cancer-(renal-pelvis−/− ureter), trophoblastic cancer,teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethralcancer, urinary system cancer, uroplakins, uterine sarcoma, uteruscancer, vaginal cancer, vulva cancer, Waldenstrom's-macroglobulinemiaand Wilms' tumor.

Kits

In another embodiment there is provided a kit or article of manufactureincluding:

-   -   an antigen binding site in the form of an immunoglobulin        variable domain, antibody, dAb, Fab, Fd, Fv, F(ab′)₂, scFv or        CDR reactive with a P2X₇ receptor, preferably a non-functional        P2X₇ receptor;    -   an immunogen for generating an immune response to a        non-functional P2X₇ receptor; and    -   a label or package insert with instructions for use in a method        described herein.

EXAMPLES Example 1 Induction of Immune Response in CML Patient

Material and Methods

Peptide

Peptide immunogen was synthesised to high purity in the formGHNYTTRNILPGLNITC to which was added the cross-linkermaleimidocaproyl-N-hydroxysuccinimide (MCS) at the C-terminal Cysresidue. The peptide was cross-linked to a carrier protein KeyholeLimpet Hemocyanin (KLH) such that the average percentage of peptide tototal peptide-protein conjugate was 40%. This peptide or the alternativepeptide GHNYTTRNILPGAGAKYYKENNVEKC (SEQ ID NO:6) similarly conjugated toKLH constituted selective epitope targets, primary and compoundrespectively that enabled differentiation of the nfP2X₇ receptors to bemade from native receptors.

Adjuvant

Imject Alum, an approved adjuvant commonly used in human immunisations,consisting of an aqueous formulation of aluminium hydroxide andmagnesium hydroxide plus inactive stabilisers in a gel, was used. Thepeptide-protein conjugate was added at a concentration of 2.5 mg/mLconjugate (1 mg/mL peptide) dropwise with thorough mixing in theadjuvant in an amount equal to 0.5 mL conjugate to 0.75 mL adjuvantcontaining 0.5 mg of target peptide epitope.

Immunisation

The immunisation schedule consisted of a primary inoculation (twoinjections subcutaneously and two injections intramuscularly) of a totalof 0.5 mg peptide followed a month later with a boost applied the sameway with 0.3 mg peptide. Serum samples were collected immediately priorto and a week post injections. Inoculation is ideally administered noless than a month after the final infusion of anti-nfP2X₇ antibody toensure no sequestration of the immunogen by residual specificanti-nfP2X₇ antibody infusate.

ELISA

Specific anti-nfP2X₇ antibody responses were measured by ELISA. Inbrief, the ELISA plate was coated with specific target peptide epitope,either naked or conjugated to a carrier other than the one against whichthe antibody response was generated, over which patient serum is addedin a descending concentration. After washing, an appropriate secondaryanti-human antibody (anti-IgM or anti-IgG types) is applied to detectand determine the concentration of specific human anti-nfP2X₇ antibodypresent in the patient serum in the form of IgM or IgG.

Following the inoculation, no IgG is detectable but a small amount ofIgM is detected. Following the boost, the IgM concentration has returnedto a baseline of zero while IgG is produced at higher concentration thanthe original IgM provided no nfP2X₇ receptor sink is present on extanttumor. In the absence of such a sink in patients for which the originaltumor has been cleared by anti-nfP2X₇ immunotherapy or alternatetherapies, a clear population of specific endogenous anti-nfP2X₇antibody is detected in the serum, of order 5 mg/kg.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

1. A method for minimising the progression of cancer in an individualwho has received a non self antigen binding site for treatment of thecancer, the method including the steps of: providing an individual whohas received a non self antigen binding site for treatment of thecancer; forming an immune response in the individual to a P2X₇ receptor;thereby minimising the progression of cancer in the individual.
 2. Themethod of claim 1 wherein the individual does not have detectable nonself antigen binding sites in circulation.
 3. The method of claim 1wherein the individual does not have detectable cancer.
 4. The method ofclaim 1 wherein the individual has developed an immune response to thenon self antigen binding site.
 5. The method of claim 1 wherein theindividual has received a non self antigen binding site that binds tonon-functional P2X₇ receptor, but not to functional P2X₇ receptor, fortreatment of the cancer.
 6. The method of claim 1 wherein immuneresponse is formed by providing an immunogen in the individual in theform of a P2X₇ receptor, or a fragment of a P2X₇ receptor that iscapable of inducing an immune response to a P2X₇ receptor in theindividual.
 7. The method of claim 6 wherein the non self antigenbinding site received by the individual for treatment of the cancer doesnot bind to a P2X₇ receptor, or a fragment of a P2X₇ receptor.
 8. Themethod of claim 6 wherein the immunogen is provided in an initialadministration to the individual, thereby forming a response thatincludes IgM production in the individual.
 9. The method of claim 6wherein the immunogen is provided in an initial administration to theindividual, thereby forming a response that includes IgM production, andat a later time, in a further administration to the initialadministration, thereby forming a response that includes IgG production.10. A process for forming a humoral immune response to cancer-associatedP2X₇ receptors in an individual who has received an anti-cancer antigenantibody for therapy of cancer, including the steps of: forming animmune response in the individual to an immunogen in the form of acancer-associated P2X₇ receptor or fragment thereof; wherein the immuneresponse is formed in the individual at a time where anti-cancer antigenantibody administered for treatment of cancer is at a level orconcentration that is substantially undetectable in the individual;and/or the humoral immune response to cancer-associated P2X₇ receptorsis formed according to an immunisation schedule whereby the amount ofantibody formed in the individual to cancer-associated P2X₇ receptor isabout 0.1 to 25 mg kg individual.